CN102159219A - Method of enhancing tgf-beta signalling - Google Patents
Method of enhancing tgf-beta signalling Download PDFInfo
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- CN102159219A CN102159219A CN2009801363692A CN200980136369A CN102159219A CN 102159219 A CN102159219 A CN 102159219A CN 2009801363692 A CN2009801363692 A CN 2009801363692A CN 200980136369 A CN200980136369 A CN 200980136369A CN 102159219 A CN102159219 A CN 102159219A
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Abstract
The present invention is directed to a method of enhancing TGF-ss signaling in a subject comprising administering to said subject a clathrin-dependent endocytosis inhibitor in an amount sufficient to enhance TGF- ss signaling. In another aspect, the invention is directed to a method of treating a condition associated with decreased TGF- ss signaling in a patient in need thereof comprising administering to said patient a clathrin-dependent endocytosis inhibitor in a therapeutically effective amount.
Description
Related application
This application requires the priority of the U.S. Provisional Application submitted to for No. 16 in JIUYUE, 2008 number 61/097,302.The whole teaching religious doctrine of the above application is as the application's reference.
Government supports
This invention, all or part of, accept HL087463 of NIH and AR052578 and subsidize.
Background of invention
Transforming growth factor (TGF β) is family's series of a pleiotrophic cytokine, suppresses the growth of most cell types (comprising epithelial cell, endotheliocyte and lymphocyte).In mammal, transforming growth actor series comprises transforming growth factor-beta 1 ,-β 2 and-β 3.Transforming growth factor-beta is the synthetic and sedimentary a kind of stimulant of extracellular matrix effective or the strongest in the body.It plays the part of important role at wound healing and tissue fibering.According to the tissue of inquiring into research, it has antiinflammatory and short scorching different activity.
In its antiphlogistic activity, the transforming growth factor-beta suppressor T cell, the B cell, macrophage and NK cell activity also suppress expression (Piccirillo etc., (1998) J.Immunol.161, the 3950-3956 of several short scorching genes at these cells; Prud ' homme and Piccirillo, (2000) J.Autoimmun.14,23-42; Li, M. etc., (2006) Biochem.Biophys.Res.Commun.344,701-707).Because it has the growth of adjusting, antiinflammatory and immunoregulatory activity, transforming growth factor-beta has been confirmed as treating the target of some diseases.For example, the transforming growth factor-beta somatic cell gene therapy has been proved to be can be used on and has prevented autoimmune disease (Piccirillo etc., (1998) J.Immunol.161:3950-3956) on the non-obese diabetes mice.The transforming growth factor-beta 1 injection also proves, on mice, can prevent the inductive arthritis (Kuruvilla etc. of collagen protein, (1991) PNAS 88,2918-2921) with the experimental allergic encephalomyelitis (Johns etc. that recur, (1991) J.Immunol 147,1792-1796), these two kinds of diseases are respectively the model of rheumatic arthritis model and multiple sclerosis.The transforming growth factor-beta injection also can prevent the reaction of allograft rejection.Transforming growth factor-beta also has been proved to be the key player (Wallick etc. that play the part of a protective in the pulse atherosclerosis of cardiovascular system in addition, (1990) J.Exp.Med.172,1777-1784). (1995) (Grainger D.J. (2004), Arterioscler.Thromb.Vasc.Biol., 24,399-304; Metcalfe and Grainger (1995), Biochem.Soc.Trans.23,403-406).For example, it has been proved to be the rising cholesterol and causes an atherosclerotic mechanism to be because a large amount cholesterol makes vascular cell reduce (Chen etc., (2007) J.Cell Sci.120,3509-3521 to the reaction of transforming growth factor-beta; Chen etc., (2007) J.Cell Sci.120,3509-3521).Transforming growth factor-beta is played the part of the role of another one complexity in canceration in addition.Early stage in canceration, this cytokine is considered to have the inhibition tumor promotion, but the stage afterwards, and its tumors inhibition activity can be lost, become on the contrary a tumor growth promoter (Derynck etc., (2001) J.Nat.Genet 29,117-129); Piek and Roberts (2001) Adv.Cancer Res.83,1-54; Stover etc., (2007), J.Cell Biochem.101,851-861).Send because reduce the transforming growth factor-beta signal, and disease that causes or situation comprise Alzheimer ((Tesseur etc., (2006) J Clin Invest.116,3060-3069; Das etc., (2006) J.Clin.Invest.116:2855-2857), systemic lupus erythematosus (sle) ((Ohtsuka etc., (1998) J.Immunol., 160:2539-2545), chronic wounds (Kim etc., (2003) .J.Cell.Phys., 195 (3): 331-6), chronic obstructive pulmonary disease (Baraldo etc., (2005) Thorax 60:998-1002), inflammatory bowel disease ((Fiocchi etc., (2001), J.Clin.Invest.108 (4): 523-526), Guillain Barre syndrome (Creange etc., (1998), J Neurol Neurosurg Psychiatry 64:162-165), and cancer of colon (Markowitz etc., Science 268,1336 (1995), Valle etc., (2008) Science:321,1361).
Owing to transforming growth factor-beta can be used for treating several diseases and improve health status, development confirms that the change agent that some signals that can strengthen transforming growth factor-beta send or the increase cell reacts transforming growth factor-beta will be useful.
Summary of the present invention
The present invention is based on a surprising discovery, and the inhibitor that clathrin relies on (clathrin-dependent) endocytosis can strengthen the transmission of transforming growth factor-beta signal.Shown in the example 1, the endocytosis inhibitor that relies at clathrin improves the inductive signal transmission of transforming growth factor-beta (being phosphorylation and the nuclear localisation that transforming growth factor-beta stimulates Smad2).
The present invention relates to a kind of transforming growth factor-beta that improves in the method that signal sends, comprise the inhibitor of the endocytosis of injecting the clathrin dependence, its quantity is enough to strengthen the transforming growth factor-beta signal and sends.
In yet another aspect, this invention is at a kind of Therapeutic Method, is applied to because reduce the signal of transforming growth factor-beta and sends the patient who is associated, the endocytosis inhibitor that a kind of clathrin for the treatment of effective dose relies on.
In yet another aspect, this invention is at a kind of Therapeutic Method, is applied to the endocytosis inhibitor that the clathrin of the patient effective dose of canceration relies on.
At further example, the present invention is that a kind of pharmaceutical composition comprises a kind of endocytosis inhibitor and a kind of HMG-CoA reductase inhibitor that effective dose net form albumen relies on for the treatment of.
The brief description of preferred embodiment picture
Above-mentioned and other object and this characteristic feature of an invention and advantage can be clearly from the following example that this is described more specifically.Being described in more detail with reference to the accompanying drawings wherein.Picture does not have certain specification, but focuses on the principle of invention.
Fig. 1 is presented at the Mv1Lu cell, the endocytosis inhibitor that clathrin relies on can improve transforming growth factor-beta 1 stimulate the phosphorylation of the Smad2 that causes and appraise and decide the position (A-I, K).The first cyclodextrin β-CD (A of these cells with 10 mg/ml, K), the MDC of 20 μ M (B), the Monensin of 40 μ M (C), the sucrose (K) of the TFP of 20 μ M (D) and 0.45M or (as shown in the figure) of variable concentrations, cyclodextrin (β-CD) (E) and TFP (F), monensin (G), (chloroquine) chloroquine (H), dynasore (I), handled 1 hour at 37 ℃, use 100pM transforming growth factor-beta 1 (add or do not add) to stimulate the transforming growth factor-beta 1 (K) of (A to I) or variable concentrations (as shown in the figure) then.37 ℃ of times (as shown in the figure) that process is different, cell pyrolysis liquid carries out 7.5%SDS to be analyzed, and west ink dot method is analyzed, and analyzes with the antibody of P-Smad2 and Smad2 and chemiluminescence development (a or top) and density quantitative (b or bottom).The expression that P-Smad2 is relative is represented (AU) A with arbitrary unit, B, C, D, K) (b) in the level relatively of the P-Smad2 of transforming growth factor-beta cell individual processing as 100% (bottom E to I).Data show is the representative of 3 independent experiments.(J) cell is handled (c with the β-CD of 10 mg/ml at 37 ℃ earlier, d), after 1 hour, these cells stimulate (b with the 100pM transforming growth factor-beta, d) do not stimulate (a) with there being transforming growth factor-beta, at 37 ℃, after 30 minutes, cell is fixed and with the antibody of P-Smad2, carries out indirect IF staining.Amplification is 200 times.Data show is the representative of 3 independent experiments.
The endocytosis inhibitor that Fig. 2 show grid albumen relies on is strengthened SARA and T β R-I co-exists in and be accumulated in the Mv1Lu cytoplasma membrane.Cell is used the TFP with 20 μ M earlier, the β-CD of 10 mg/ml, and (chloroquine) choloquine (CQ) of the dynasore of 40uM (Dyn) or 200 μ M handles, and 37 ℃, 1 hour.The cell of handling stimulates with the transforming growth factor-beta 1 of 100pM then or does not add transforming growth factor-beta 1.Through 30 minutes under 37 ℃, cell is fixed and with T β R-I, the antibody of SARA carries out indirect IF staining (little Fig. 1-10 shows T β R-I) and SARA (little Figure 11-20), DAPI (nuclear staining) (little Figure 21-30).Show the dyeing (little Figure 31-40) after merging.Illustration (in little Figure 33-36) shows that SARA and T β R-I co-exist in and be accumulated in cell membrane.
Fig. 3 is presented at the Mv1Lu cell, and the dependent endocytosis inhibitor of clathrin increases the expression that transforming growth factor-beta stimulates PAI-1.(A-G) cell handle earlier variable concentrations (as shown in the figure) β-CD (A, C), thioridazine (B), TFP (D), chloroquine (chloroquine) (E), monensin (monensin) (F) and MDC (G) is following 1 hour at 37 ℃,, stimulate with the 50pM transforming growth factor-beta 1 then.At 37 ℃, through 2 hours, in the cell pyrolysis liquid expression of the mRNA of PAI-1 and G3PDH by northern hybridization analysis (on) and use PhosphoImager quantize (descend) (A, B) or real-time RT-PCR detection (C-G).Experiment is triplicate to be implemented.What show the table data is the representative of 3 independent experiments.The expression data of the PAI-1 that does not have transforming growth factor-beta to stimulate be as 100% (A, B) or 1 times (C is to G).Experiment has been carried out triplicate altogether.* show that the matched group that numeric ratio unrestraint agent is handled has significant statistical difference, p＜0.001.* shows that numeric ratio unrestraint agent processing matched group has significant statistical difference, P＜0.05.(H) cell was handled 1 hour at 37 ℃ with the dynasore of variable concentrations (as shown in the figure) earlier, stimulated with the 50pM transforming growth factor-beta 1 then.Through 2 hours at 37 ℃, with the mRNA of the quantitative PAI-1 of real-time RT-PCR.With the transforming growth factor-beta 1 individual processing as 1 (100%).Experiment has been carried out the same form three times altogether.This data presented is 3 independently representatives of experiment.
Fig. 4 has shown that the coronary tissue credit of the mouse that wild type and ApoE are invalid analyses (A) and T β R-II immunofluorescence dyeing (B).The mouse that wild type and ApoE are invalid, do not have dynasore handle (DMSO solvent a, c) or 1 milligram of dynasore/ kg body weight (b d) handles and once carried out for 8 weeks altogether in per two days.H﹠amp is analysed in the coronary tissue credit; E dyes (A) and carries out indirect IF staining analysis (B) with T β R-II antibody.The position of asterisk (*) indication coronary artery tube chamber.This arrow is illustrated in the immunostaining that aorta inner skin has T β R-II.
Detailed description of the invention
Preferred embodiment of the present invention is described as follows.
In this clause, " one " or " one " is meant to comprise one or more, unless otherwise prescribed.
The present invention be directed to and improve the reaction method that the transforming growth factor-beta signal sent or increased transforming growth factor-beta, comprise the inhibitor that relies on endocytosis with a kind of clathrin.The present invention also at various diseases or health status with by strengthening the reaction that the transforming growth factor-beta signal sent or increased transforming growth factor-beta.Award treatment or improvement.
Term used herein " transforming growth factor-beta " comprises transforming growth factor-beta 1 and transforminggrowthfactor-, the mutant of transforming growth factor-beta 3 and transforming growth factor-beta (shows similar biological activity and is different from the proteic simple or a plurality of sudden changes of natural transforming growth factor-beta as transforming growth factor-beta albumen, for example change, increase or omit one or above aminoacid).When blood plasma transforming growth factor-beta level increases or the signal of transforming growth factor-beta reaction sent when increasing when a cell, the transforming growth factor-beta signal sends and can strengthen.The level of transforming growth factor-beta can be different under different condition in the blood plasma.For example, when the expression of transforming growth factor-beta gene in histiocyte increases or the decomposition of transforming growth factor-beta when being suppressed, the transforming growth factor-beta level can increase in the blood plasma, strengthens the transmission of transforming growth factor-beta signal a cell is increased the reaction of transforming growth factor-beta.For example, by the phosphorylation that transforming growth factor-beta stimulates Smad2, clathrin dependence (chatherin-dependent) endocytosis inhibitor can strengthen cell transforming growth factor-beta is reacted.Eukaryotic cell utilizes clathrin to rely on and ind two endocytic pathway.The characteristics that clathrin relies on endocytosis have three phases: the formation of coated pit (formation of the coated pit), the hoop of cave in (invagination of the coated pits) and the coated vesicle of coated pit break (pinching off of the coated vesicles) (Mousavi etc., 2004).Here so-called one " the endocytosis inhibitor that clathrin relies on " is that a change agent can utilize any mechanism to suppress clathrin dependence endocytosis.According to the present invention, the change agent that can be used for suppressing the endocytosis that clathrin relies on is: and methyl-beta-schardinger dextrin-(β-CD), hydrophobic amines is (as phenothiazine (phenothiazines), monodansylcadaverine and chloroquine (chloroquine)), monensin (monensin), height ooze sucrose (hyperosmotic sucrose) and dynasore.Phenothiazine (Phenothiazines) includes but not limited to, chlorpromazine (chlorpromazine), fluphenazine (fluphenazine), mesoridazine, perphenazine (perphenazine), prochlorperazine, promazine, thioridazine (thioridazine), trifluoperazine (trifluoperazine) and triflupromazine.β-CD suppresses the endocytosis that clathrin relies on by the cholesterol of removing cytoplasma membrane selectively.The function that hydrophobic amines influences clathrin and clathrin coated vesicle suppresses the endocytosis that clathrin relies on.Monensin suppresses the endocytosis that clathrin relies on by dissipation proton gradient (proton gradient).Height oozes sucrose by preventing that clathrin and adapter (adaptors) reciprocal action from suppressing the endocytosis that clathrin relies on.Dynasore suppresses dynaminGTPase, this dynamin GTPase inhibitor to coated pit formation favourable.
In one embodiment, the endocytosis inhibitor of clathrin dependence is that the inhibition coated vesicle is disconnected from the hoop of cytoplasma membrane.In another embodiment, the endocytosis inhibitor that relies on of clathrin be can irritation cell PAI-1 expression (PAI-1 is the gene of a cell to the transforming growth factor-beta stimuli responsive).In another embodiment, dynasore itself is the endocytosis inhibitor that clathrin relies on, and itself has the activity of performance transforming growth factor-beta, and it also is the parody or the mimicry body of transforming growth factor-beta.
In certain embodiments, the endocytosis inhibitor of clathrin dependence is a kind of dynaminGTPase inhibitor.In further embodiment, dynasore itself is a dynamin GTPase inhibitor.Dynasore has the chemical constitution of formula (I):
The present invention comprises that also a kind of method is used for treating owing to the transforming growth factor-beta signal sends the disease that minimizing causes.The transforming growth factor-beta signal sends and reduces diseases associated, includes but not limited to inflammation disease, autoimmune disease, cancer, cardiovascular diseases and some dermatosis.Inflammation disease includes but not limited to, multiple sclerosis (multiple sclerosis).In certain embodiments, inflammation disease is that the combination of a group disease comprises Alzheimer (Alzheimer ' s disease), asthma (asthma), chronic obstructive pulmonary disease (chronic obstructive pulmonary disease), multiple sclerosis (multiple sclerosis), rheumatoid arthritis (rheumatoid arthritis), atherosclerosis (atherosclerosis), the Guillan-Barre syndrome, systemic lupus erythematosus (sle) (systemic lupus erythematosus).At another example, atherosclerosis is a kind of because the symptom that inflammation causes.
On the one hand, the present invention is an a kind of method for the treatment of in the cardiovascular disease, the inhibitor of the endocytosis that relies on comprising a kind of clathrin of giving a treatment of patient effective dose.
The present invention is also at a method for the treatment of a kind of skin symptom.This symptom is the abnormality proliferation and/or the growth of relevant skin histology, the disease of example is psoriasis (psoriasis), basal cell carcinoma (basal cell carcinoma), squamous cell carcinoma (squamous cell carcinoma), keratoacanthoma (keratoacanthoma), actinic keratosis (actinic keratosis), the chain ichthyosis of X (X-linked ichthyosis), acne (acne), dermatitis (dermatitis) and keratinization of epidermis be (epidermolytic hyperkeratosis) excessively.
The present invention also is used for the treatment of cancer at the another one method, the inhibitor of the endocytosis that relies on comprising a kind of clathrin of giving a treatment of patient effective dose.Exemplary tumor comprises malignant tumor (malignant tumors), adenocarcinoma (adenocarcinomas), cancer (carcinomas), sarcoma (sarcomas), malignant tumor (malignant neoplasms), leukemia (leukemias) and epithelial cell source cancer (epithelial cell derived).The derive example of cancer of epithelial cell includes but not limited to: breast carcinoma (breast cancer), colon cancer (colon cancer), ovarian cancer (ovarian cancer), pulmonary carcinoma (lung cancer) and carcinoma of prostate (prostate cancer).At another example, this method relates to from the treatment of the state of an illness and comprises melanoma (melanoma), myeloma (myeloma), and lymphoma lymphoma forms, and comprises the inhibitor of the endocytosis that relies on to a kind of clathrin.Cancer by the inventive method processing.Can comprise any source such as chromosomal abnormality (chromosomal abnormalities), growth of degeneration (degenerative growth) and retrogressive development obstacle (developmental disorders), mitogenic agent (mitogenic agents), ultraviolet radiation (UV), viral infection (viral infections), oncogene (oncogenes), mutant gene (mutations in genes), gene is in the unsuitable expression of cell, or carcinogen.
In another embodiment, The present invention be directed to the inhibitor that a kind of Therapeutic Method comprises the endocytosis that a kind of clathrin to one of patient treatment effective dose relies on.The feature of this symptom is relevant to growth factor-beta signal transmission minimizing.Wherein characteristics are that transforming growth factor-beta signal transmission minimizing is not to be caused by infection involving chlamydia pneumoniae (Chlamydia pneumoniae infection).
In other embodiments, The present invention be directed to a kind of Therapeutic Method and comprise that the inhibitor of the endocytosis that a kind of net form albumen to one of patient treatment effective dose relies on suppresses canceration.Suppress canceration and comprise generation or the precancerous condition or slow that prevents relevant cancer, stop or reversing the cancer or the precancerous condition of deterioration.Precancerous condition comprises, for example, hypertrophy (hyperplasia), change and give birth to (metaplasia), dysplasia (dysplasia), oral leukoplakia (oral leukoplakia), photochemical keratinization (actinic keratosis) solar energy keratinization (solar keratosis)), the precancerous polyp of colon cancer or rectal cancer (precancerous polyps of the colon or rectum), Weishang skin atypical hyperplasia (gastric epithelial dysplasia), adenoma sexual abnormality (adenomatous dysplasia), hereditary nonpolyposis colon cancer syndrome (HNPCC) (hereditary nonpolyposis colon cancer syndrome (HNPCC)), Barrett esophagus (Barrett ' s esophagus), bladder dysplasia (bladder dysplasia), (precancerous cervical) situation before the cervical cancer.
In yet another aspect, this invention is that a kind of method for the treatment of cardiovascular disease comprises the endocytosis inhibitor that relies on to a kind of clathrin for the treatment of effective dose of patient therein.In yet another aspect, The present invention be directed to a kind of Therapeutic Method and treat cardiovascular disease to the inhibitor of the endocytosis of a kind of clathrin dependence of a treatment of patient effective dose.
The present invention be directed to a kind of Therapeutic Method and comprise that the dynasore to a treatment of patient effective dose treats cardiovascular disease.At an embodiment, cardiovascular disease is exactly an atherosclerosis.Another example, the present invention also comprises the inhibitor that gives 3-hydroxy-3-methyl CoA-reductase (HMG-CoA reductase).At further example, the HMG-CoA reductase inhibitor is a kind of statins (statin).Statins comprises lovastatin model (lovastatin), simvastatin (simvastatin), pravastatin (pravastatin), fluvastatin (fluvastatin), atorvastatin (atorvastatin), itavastatin (itavastatin), rosuvastatin (rosuvastatin) and rivastatin (rivastatin).The present invention is also at comprising suffering from the risk patient of myocardial infarction, gives endocytosis inhibitor that a kind of clathrin relies on to prevent myocardial infarction.Have the patient of high-level low density lipoprotein, LDL (LDL) to have the risk of suffering from myocardial infarction, patient can suffer atherosclerotic misery.
In this clause, " treatment " or " therapy " comprises the disease symptom that prevents or delay a kind of disease, complication, or biochemical labelling, alleviate or mitigation symptoms or prevention or inhibition further develop disease, situation or disease.One " treatment effective dose " is an amount, wherein combines separately or with one or more other activating agents, can control, and reduces, and suppresses, and improvement prevents or others influence one or more symptom diseases or situation is treated.In this clause, the effective dose of the treatment of the endocytosis inhibitor of one one clathrin dependence is to be enough to improve the transforming growth factor-beta signal send.
The present invention also comprises a kind of pharmaceutical composition, comprises that the endocytosis of a kind of clathrin dependence of a treatment effective dose suppresses inhibitor and HMG-CoA reductase inhibitor.In one embodiment, the endocytosis inhibitor of clathrin dependence is dynasore itself.In another embodiment, the HMG-CoA reductase inhibitor is a statins (statin).
Pharmaceutical composition comprises a kind of at pharmaceutically acceptable carrier or excipient.At pharmaceutically acceptable carrier or excipient is to select according to the route of administration of expecting in treatment.The approach of administration depends on how to handle disease.For example, therapy system disease such as leukemia or lymphoma may be first-selected intravenous injections, and treatment gastrointestinal system disease first-selection is oral.A skilled technician can determine instructions of taking and consumption according to the anti-ying relation of standard consumption, avoids unnecessary laboratory facilities.When making decision, must consider that the correlation circumstance of handling comprises one or multiple situation, the selection of medicine, age, the reaction of body weight and individual patients and patient's severity of symptom.
Therefore, according to different situations, medicine can be oral, non-intestinal (intestinal is outer), and collunarium, vagina, rectally, tongue, sublingual administration, cheek ground (buccally), intrabuccaly and subcutaneous (transdermal) give patient.Therefore, the design oral cavity, tongue, the Sublingual, the medicine that cheek, intrabuccal are taken can utilize the method for having known, avoids unnecessary laboratory facilities, for example uses inert diluent or and edible carrier.This medicine can be included in gelatine capsule or compression is in blocks.For the medicine of oral medication, the composition of medicine of the present invention can merge and be used for the form of tablet, lozenge, capsule, medicated wine, suspension, syrup, chip, chewing gum and similar product with excipient.
Tablet, pill, capsule, lozenge and similar product also can contain binding agent, adjuvant agent, disintegrating agent, lubricant, sweeting agent and flavoring agent.The example of some binding agents comprises microcrystalline Cellulose (microcrystalline cellulose), gum tragacanth (gum tragacanth) or gelatin (gelatin).The example of adjuvant agent comprises starch or lactose.Some examples of disintegrating agent comprise alginic acid, corn starch or the like.The example of lubricant comprises: magnesium stearate or potassium stearate.The example of a fluidizer is a colloid silicon carbon dioxide.Some examples of sweeting agent comprise sucrose, glucide and similar product.The example of flavoring agent comprises Herba Menthae, methyl salicylate, orange fragrance or the like.
Composition of the present invention can absorb outward via intestinal, for example, by intravenous injection, intramuscular injection, intrathecal injection or subcutaneous injection.Intestinal is outer can be by integrating solution or the suspension of consisting of of the present invention.This solution or suspension also can comprise aseptic diluent such as injection water, normal saline, fixing oil, Polyethylene Glycol, glycerol, propylene glycol or other synthetic.Also may comprise in the outer prescription of intestinal, for example, antibacterial benzyl alcohol or methyl benzoic acid ester, antioxidant, for example ascorbic acid or sodium sulfite, chelating agen such as EDTA.Buffer agent, as acetic acid, citric acid or phosphate and adjustment agent also can add as sodium chloride or glucose.The intestinal external preparation can be packaged in ampoule, the bottle that disposable syringe or multiple dose glass or plastics are made.
Rectally comprises to the pharmaceutical composition medicine through rectum or large intestine.This can use suppository or coloclysis.Suppository can known at an easy rate method preparation.For example, can add hot glycerine,, can add pure water after adding the hot glycerine mixing, pour the mixture mould into and make suppository to be dissolved in the pectin composition in the glycerol to about 120 ℃.
Transdermal administration comprises that percutaneous absorbs percutaneous composition.Preparation capable of permeating skin comprises patch, ointment, cream, gel, ointment or the like.
Following Example further specifies the present invention, but should not limit its scope with any explanation.
Example 1: clathrin relies on the endocytosis inhibitor and strengthens the transforming growth factor-beta letter
Number send and reaction
Summary
The endocytosis that clathrin relies on is considered to relevant with the reaction of transforming growth factor-beta irritation cell, but the inductive signal of transforming growth factor-beta be sent in where take place in the cell still unclear.Our explanation here, the endocytosis inhibitor that clathrin relies on can stop to be coated with the endocytosis of hole developmental stage, suppress the internalization and the common location and the accumulation that promote T β R-I and SARA at cell membrane of cell surface bind to transforming growth factor-β, these effects are well-known.These inhibitor can improve the inductive signal of transforming growth factor-beta and send (such as the phosphorylation of Smad2/the appraise and decide expression of position and PAI-1).And dynasore is a new dynamine GTPase activity inhibitor of determining, in all tests, it is a kind of effective inhibitors, also is a kind of effective transforming growth factor-beta reinforcing agent.And dynasore itself can improve the atherosclerosis of the vascular endothelial cell of the invalid mouse of the ApoE of hypercholesterolemia, because it can offset the reaction sensitivity of hypercholesterolemia compacting transforming growth factor-beta.This effect believes that influencing transforming growth factor-beta by it sends in vascular cell endocytosis and signal.
Transforming growth factor-beta is family's series of a pleiotrophic cytokine.In mammal, transforming growth actor comprises transforming growth factor-beta 1 ,-β 2 and-β 3.This is a growth regulator with dual-use function
1It suppresses the most cell types growth, comprises epithelial cell, endotheliocyte and lymphocyte, and it can stimulate the growth of Interstitial cell.The activity of the growth regulating of transforming growth factor-beta is with carcinogenecity, immunomodulating and cell differentiation implication.Transforming growth factor-beta is the synthetic and sedimentary stimulant of known effective cell epimatrix.It plays the part of important role at wound healing and tissue fibering.The tissue that depends on research, transforming growth factor-beta have antiinflammatory and short scorching activity.Because the activity of its anti-inflammatory and immunity, transforming growth factor-beta is the cytokine 2,3 of a protectiveness in the atherosclerosis of cardiovascular system.
The stimulating activity of transforming growth factor-beta is to form heterooligomeric transform growth factor-beta receptor complex because it can be induced at cytoplasma membrane
4,5In the inside of this receptor complex, the II transforming growth factor-beta receptor of primary activity (T β R-II) activates I transforming growth factor-beta receptor (T β R-I) phosphorylation.This activates and the I transforming growth factor-beta receptor (T β R-I) of phosphorylation can phosphorylation Smad2 and Smad3.This phosphorylation can be promoted by Smad albumen grappling receptor (SARA)
6,7The Smad2/Smad3 of phosphorylation is transported to and is accumulated in the nucleus with the Smad4 combination.In nucleus, their conditioned reaction gene transcription.It is a negative regulator that Smad7 sends the transforming growth factor-beta signal.It is to follow with Lipid Rafts (lipid rafts)/cytoplasma membrane microcapsule (caveolae) to combine, and transforming growth factor-beta/transform growth factor-beta receptor decomposes
8,9Cell is to be chosen in the path that clathrin dependency and cytoplasma membrane microcapsule rely on endocytosis by transforming growth factor-beta to decide to the reaction of transforming growth factor-beta,
8-12The former promotes signal to send and cell effect, and the latter then causes the rapid decomposition of transforming growth factor-beta/transform growth factor-beta receptor and reduces transforming growth factor-beta cell effect sensitivity
8-12Though the endocytosis that clathrin relies on is to participate in signal to send
3,6-15, wherein the signal transmission path in the transforming growth factor-beta inducing cell is still unclear
16
Endosome (Endosome) is considered to the important actuator (mediator) that the inductive signal of transforming growth factor-beta sends
3,8-15This can suppress transform growth factor-beta receptor internalization and the inductive cell effect of transforming growth factor-beta based on observing overexpression dynamin negative mutant K44A, also has SARA and interior body tag to be positioned at endosome altogether
8,13But, Lu et.al
16Be shown to overexpression dynamin K44A expression inhibiting transform growth factor-beta receptor interiorization, the inductive signal of transforming growth factor-beta sends and its cell effect but do not change.Send and the contradictory result of the induced cell effect role of transforming growth factor-beta about endosome transforming growth factor-inductive signal a bit, this is because different dynamin negative mutant K44A
8Expression different with because the different result of experimental system of usefulness
8,16Define the cell site of determining that the inductive signal of transforming growth factor-beta sends, we comprise dynamin inhibitor dynasore with the endocytosis inhibitor of the clathrin dependence of several known action
17,18, study these and send and the transforming growth factor-beta cell effect about the inductive signal of endosome transforming growth factor-beta.The method is to avoid producing because of the difference of the expression of dynamin K44A the alternative of Different Results.
There are some chemical compounds to be proved to be able to suppress clathrin and rely on endocytosis.These comprise methyl-beta-schardinger dextrin-(β-CD)
19, phenothiazine
20-22, monodansylcadaverine (MCD)
23, chloroquine
24, monensin
25, height oozes sucrose and oozes
26And dynasore
17,18β-CD suppresses the endocytosis that clathrin relies on by selective extraction cell membrane cholesterol.Hydrophobic amine is phenothiazine for example, and the function that chloroquine and MDC etc. influences clathrin and clathrin coated vesicle suppresses the endocytosis that clathrin relies on
20,24Monensin is monovalent ionophore, suppresses the endocytosis that clathrin relies on by the dissipation proton gradient
25Height oozes sucrose by preventing that clathrin and adapter (adaptors) reciprocal action from suppressing the endocytosis that clathrin relies on.And, dynasore itself be one can permeation cell the active inhibitor of dynamin GTPase, this dynaminGTPase inhibitor the process of endocytosis to coated pit formation favourable
17,18
As previous report
6-15If the transforming growth factor-beta signal is sent in the endosome and takes place, the inhibitor of the endocytosis that clathrin relies on estimates to weaken the location of the signal transmission of transforming growth factor-beta as Smad2 phosphorylation and nuclear
4,5In order to test this theory, the Mv1Lu cell is used or is handled at 37 ℃ with the endocytosis inhibitor that clathrin relies on, and 30 minutes, uses then or 100pM transforming growth factor-beta useless processing.In several periods, use P-Smad2 and Smad2 antibody, western blot analysis analysis of cells P-Smad2 and Smad2.As shown in Figure 1, β-CD, MDC, monensin and trifluopromazine (TFP) can both make transforming growth factor-beta be excited the Smad2 phosphorylation to strengthen its enhanced mode relevant with the time (being respectively Figure 1A a, 1Ba, 1Ca and 1Da).If stimulated 60 minutes with transforming growth factor-beta, β-CD, TFP, MDC and monensin, strengthen transforming growth factor-beta be excited Smad2 phosphorylation～1.5-2.5 doubly (be respectively Figure 1A b, 1Bb, 1Cb, 1Db).For further investigation, these inhibitor are to the influence of Smad2 phosphorylation, and cell, was handled 30 minutes earlier at 37 ℃ at the inhibitor of variable concentrations, and then with the transforming growth factor-beta of 100pM 37 ℃ of stimulations after 30 minutes, the Cell sap western blot analysis.Shown in Fig. 1 E to 1I, β-CD, TFP, monensin (monensin), chloroquine (chloroquine) and the enhanced transforming growth factor-beta of dynasore are excited the phosphorylation of Smad2 and are strengthened along with the increase of concentration.With the β-CD of 10 mg/ml, TFP (24 μ M), monensin (40 μ M), chloroquine (200 μ M) and dynasore (40 μ M) strengthen transforming growth factor-beta and are excited Smad2 phosphorylation～2-3 doubly.In order to determine the effect of β-CD on the transforming growth factor-beta excited nucleus location of P-Smad2, the Mv1Lu cell earlier with or without β-CD (10 mg/ml) at 37 ℃, handled one hour, reuse transforming growth factor-beta (10pM) was handled 30 minutes, and the fluorescence staining analysis P-Smad2 that reuse is indirect appraises and decides the position.Shown in Fig. 1 J, β-CD and transforming growth factor-beta promote the position of appraising and deciding of P-Smad2 (d) together, however there be appraise and decide (b and a c) who does not just promote P-Smad2 alone in each.
Here Ce Shi endocytosis inhibitor can suppress the hoop disconnected (formation of endosome) of endocytic vesicle from plasma membrane, and make its hole stage that rests on coating, and stop the endocytosis process
17-26This shows that to the transmission of transforming growth factor-beta signal, the stage in the hole of coating, it played the part of an important role in the endocytosis process that clathrin relies on.It is important sending at the transforming growth factor-beta signal for the hole stage that will determine to be coated with, and we are with the high sucrose (0.45M) that oozes or β-CD processing Mv1Lu cell, reexamines these transit cell growth factor-betas and is excited the phosphorylation of Smad2.The formation of the coated pit (coated pit of Class1) that the sucrose that height oozes is considered to suppress shallow or the cluster of receptor
26In the endocytosis process that clathrin relies on, β-CD is considered to suppress from the progress of shallow coated pit (Class1 coated pit) with opisthotonos coated pit (type 2 coated pits)
19Shown in Fig. 1 K, handle cell in transforming growth factor-beta with variable concentrations, the sucrose that height oozes can suppress transforming growth factor-beta and be excited the phosphorylation (Fig. 1 Ka and 1Kb) of Smad2.In 100pM transforming growth factor-beta stimulated cells, the sucrose that height oozes weakens Smad2 phosphorylation～60% (Fig. 1 Kb).On the other hand, in the transforming growth factor-beta of variable concentrations, β-CD can strengthen TGF-β 1 really and be excited the phosphorylation of Smad2.And have β-CD to handle not compare, β-CD can strengthen transforming growth factor-beta and be excited Smad2 phosphorylation～2-4 doubly (Fig. 1 Ka and 1Kb).These results advise that the transforming growth factor-beta signal is sent in the coated pit stage generation of Class1.
In T β R-I-SARA-Smad2 coordination compound, by the combination of inducing T β RI and SARA (as the deadman that is used for Smad2), the combination of SARA and Smad2, T β RI makes the phosphorylation of Smad2, causes transforming growth factor-beta to stimulate the Smad2 phosphorylation
6,7If strengthening TGF-β-signal, the endocytosis inhibitor that clathrin relies on sends (transforming growth factor-beta-be excited Smad2 phosphorylation), be by increasing the accumulation of T β R-I/T β RII coordination compound at coated pit, they should promote T β RI and SARA common localization and the accumulation at plasma membrane.In order to test this theory, the Mv1Lu cell is stimulated by the 100pM transforming growth factor-beta.At 37 ℃, 30 minutes, it handled cell with inhibitor once more, re-used the antibody of T β RI and SARA, carried out the microscopy analysis of fluorescence immunoassay method of inspection.Fig. 2 shows, at the Mv1Lu cell, transforming growth factor-beta stimulates T β RI and SARA common localization and the accumulation (Fig. 2, panel 32 counter plates 31) at coated pit (endosome) alone.This and former report are said: transforming growth factor-beta strengthens the transform growth factor-beta receptor interiorization and meets
16But, coprocessing transforming growth factor-beta and TFP, β-CD, dynasore, or chloroquine promotes T β RI and SARA in plasma membrane localization and accumulation (Fig. 2, little Figure 33-36 inserts) altogether.These endocytosis inhibitor cannot cause that separately T β RI and SARA are in plasma membrane localization and accumulation (Fig. 2, little Figure 37-40 couple of little Figure 31) altogether.These results support following notion: the endocytosis inhibitor that clathrin relies on, be by the accumulation that promotes SARA and transform growth factor-beta receptor coordination compound in the hole stage of plasma membrane or coating, send (being excited the localization of Smad2 phosphorylation and nuclear) such as transforming growth factor-beta and strengthen the transforming growth factor-beta signal.
The PAI-1 gene is that the stimulation of cellular response transforming growth factor-beta has one of gene of research most
4,5The initiated area of PAI gene contains several Smad2/3 binding sites, and these binding sites have been used as the expression that transforming growth factor-beta response assemblies (responsive element) strengthens a reporter gene (reporter gene)
4,5In order to prove that the endocytosis inhibitor that these clathrins rely on is the reinforcing agent that the transforming growth factor-beta signal sends, we study these inhibitor, at the Mv1Lu cell, and the expression that PAI-1 stimulates because of transforming growth factor-beta.Several concentration class of the β-CD of cell usefulness variable concentrations, thioridazine, TFP, MDC, monensin and chloroquine were handled 1 hour at 37 ℃, used TGF-β 1 (50pM) to stimulate together then.At 37 ℃, after 2 hours, use the rna blot analysis (Fig. 3 A and 3B) of quantity and the relative quantity that real-time reverse transcriptase PCR (Fig. 3 C-3G) is analyzed the PAI-1 messenger RNA.As shown in Figure 3, these inhibitor enhancing transforming growth factor-beta are excited to express with concentration relevant.B-CD (10 mg/ml) (A, C), thioridazine (thioridazine) (5 μ M) (B), TFP (6 μ M) (D), chloroquine (100 μ M) (E), monensin (10 μ M) (F) and (G) transforming growth factor-beta of enhanced PAI-1-be excited to express 1.8 to 4.5 times of MDC (100 μ M).Owing to find the expression that dynasore can stimulate PAI-1 alone, we are with the dynasore of several variable concentrations and have and do not have the existence of 50pM transforming growth factor-beta to get off to determine the expression of PAI-1 at the Mv1Lu cell.Show the PAI-1 that dynasore (25 μ M) stimulates alone expresses and the arrival of 50pM transforming growth factor-beta is same degree at Fig. 3 H.At 50 μ M, dynasore strengthens transforming growth factor-beta to be excited PAI-1 and to express～5 times.
Recently have been found that the transforming growth factor-beta reaction sensitivity that is compressed in the vascular cell is played the part of important role in the inductive atherosis pathogenesis of hypercholesterolemia
3,12We suppose for example endocytosis inhibitor of clathrin dependence of transforming growth factor-beta reinforcing agent, can improve because of hypercholesterolemia suppresses vascular cell transforming growth factor-beta induction sensitivity, and inductive atherosis
3,12In order to test our hypothesis, we every other day, with intraperitoneal administration (body weight of 1 milligram of dynasore/ kilogram), had for 8 weeks altogether with the invalid mouse of hypercholesteremic ApoE.We select dynasore that three reasons are arranged.These comprise: 1) utilize Mv1Lu cell and other cell type to comprise aortal endotheliocyte (BAEC cell) and the Chinese hamster ovary cell (Chinese hamster ovary celI) of cattle, among the test inhibitor, dynasore is the strongest transforming growth factor-beta reinforcing agent (inedited result).At these cell types, dynasore (50 μ M) can strengthen TGF-β be excited PAI-1 expression～4-5 doubly, 2) Dynasore is unique inhibitor, the PAI-1 that can stimulate alone expresses.This is a transforming growth factor-beta booster and transforming growth factor-beta analogies, 3) and intraperitoneal is to dynasore (1 mg/kg body weight), and per 2 days are once, after 8 weeks, detect the liver of wild type mouse, heart, lung and kidney do not have the unusual of significantly not macroscopic or microcosmic.Show, have many macrophages or foam cell to be connected to coronary artery on the endothelium (Fig. 4 Ac) the invalid mouse of ApoE as Fig. 4 A.On the contrary, the invalid mouse of hypercholesteremic ApoE of handling at dynasore (Fig. 4 Ad), seldom macrophage or foam cell (Fig. 4 Ad).Dynasore does not influence the level (mouse that the ApoE that Dyasore handled is invalid, 460 ± 50 milligrams/deciliter, to the invalid mouse of ApoE that does not have dynasore to handle, 470 ± 61 milligrams/deciliter) of the invalid mouse cholesterolemia of ApoE.In the wild type mouse with or handle without dynasore, their coronary artery shows normal morphology (Fig. 4 Aa and 4Ab).Those blood vessel cholesterol levels are respectively 120 ± 10 and 125 ± 20 milligrams/deciliter in the wild type mouse of using or handling without dynasore.
The invalid mouse of ApoE
12Aortal endothelium, hypercholesterolemia can suppress the expression of T β RII, we check in the mouse of handling with dynasore, the expression of T β RII.Shown in Fig. 4 B, with or the wild type mouse of handling without dynasore, we find to have in aortal endothelium and in the smooth muscle T β RII to dye, and represent to have the expression (Fig. 4 Ba and 4Bb) of T β RII.But, reach in our the aortal endothelium in the invalid mouse of the ApoE that does not have dynasore to handle and do not find T β RII dyeing (Fig. 4 Bc) in the smooth muscle, expression does not have the expression of T β RII.As if Dynasore handles, recover T β RII dyeing in the crown moving endothelium of those mouse (Fig. 4 Bd).Enjoyably, near the smooth muscle of our discoveries endothelium, the slight dyeing of T β RII (Fig. 4 Bd) is arranged.These results advise that dynasore is effectively to improving atherosis, the invalid mouse of ApoE, are the inhibition of T β RII being expressed owing to the aortal endothelium hypercholesterolemia of dynasore counteracting to small part
12
The inhibitor of the endocytosis that clathrin relies on is considered to be suppressed at the process of the different step of endocytosis.The sucrose that height oozes suppresses the formation of Class1 coated pit (coated pits)
19,28B-CD and dynasore suppress from the Class1 coated pit to type 2 progress
17,18Phenothiazines, MDC and chloroquine suppress the process of coated pit progress of 3 from type 2 to type
23,24Monensin and dynasore suppress coated pit proceeds to coated vesicle (coated vesicles) from type 3 process
18,25Except that height oozed sucrose, the inhibitor of all tests strengthened transmission of TGF-signal beta and response.These inhibitor stop the hole stage of the coating of endocytosis, and this shows that the induced signal of transforming growth factor-beta mainly occurs in the coated pit stage.Height oozes sucrose to be suppressed the formation of Class1 coated pit and weakens transmission of transforming growth factor-beta signal and response.Owing to cause the formation of type 2 coated pits and the process that coated vesicle is sent out formation, the two process is all wanted dynamin, and the effect of the inhibition dynamin by dynasore estimates to hinder both processes of endocytosis that clathrin relies on and the accumulation that increases coated pit.Dynasore is than other inhibitor in this explanation, phenothiazines for example, MDC, monensin, it is the reason of the more effective inhibitor of the endocytosis that relies on of clathrin,, also be stronger transforming growth factor-beta reinforcing agent, because of all inhibitor only suppress endocytosis single step.
The endocytosis inhibitor that all these clathrins rely on can suppress the interiorization (accompanying drawing 1) of the affined transforming growth factor-beta of cell surface.Show with immunofluorescence microscopy, remove height ooze sucrose, all inhibitor can strengthen T β RI and SARA localization and the accumulation at plasma membrane.This activity with report meets, and just they can stop Class1, the hole of type 2 or type 3 coatings
17The process of endocytosis
17-26Report points out that overexpression dynamin K44A can strengthen the accumulation of T β RI at plasma membrane
16Lu etc.
16Report: overexpression dynamin K44A can suppress the internalization of cell surface T β RI.But do not influence transforming growth factor-beta and be excited cell effect.In fact, in their experimental system, the overexpression of their data show dynaminK44A can strengthen transforming growth factor-beta simulated response～2 times.We think the overexpression of in the data of they K44A cells that wild type dynamin
16Not a suitable experiment control.The overexpression of wild type dynamin can strengthen transforming growth factor-beta-be excited the ever-increasing formation of cell effect (expression of 3TP luciferase and ARE-luciferase) by the hole that promotes endocytosis and coating
16The overexpression of dynaminK44A can strengthen transforming growth factor-beta and be excited the response of cell, and it is by the hole stage in coating
16Stop endocytosis.
Our recent findings: cultured cell and hypercholesterolemia ApoE invalid the aortal endothelium of mouse in, the reaction sensitivity of cholesterol compacting transforming growth factor-beta
3,12From cumulative proof, to atherosis, the transforming growth factor-beta in the blood is the cytokine of a protection
(3-7), this shows that hypercholesterolemia causes atherosis, and is to a certain extent minimum, is the reaction sensitivity by the compacting transforming growth factor-beta
3,12We here show, dynasore is a strong transforming growth factor-beta reinforcing agent, improve atherosis, general by offsetting the reaction sensitivity of suppressing transforming growth factor-beta because of hypercholesterolemia effectively the invalid mouse of ApoE
3,12Because of reducing transforming growth factor-beta level and/or transforming growth factor-beta reaction sensitivity, with other lysis implication, for example autoimmune disease
29, therefore effective transforming growth factor-beta reinforcing agent, for example dynasore or dynasore similar compounds are to be used for the treatment of such treatment of diseases chemical compound.
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The present invention is especially with showing and describe the preferred embodiment reference that the those skilled in the art knows, is not departing from the scope of the present invention under the situation that comprises additional claim, can do a little change and modification to form and details.
Claims (16)
1. one kind is used to strengthen the method that individual transforming growth factor-beta signal sends, and comprises the endocytosis inhibitor of the clathrin dependence that gives an individual q.s, sends to strengthen the transforming growth factor-beta signal.
2. the method for claim 1, wherein the endocytosis inhibitor of clathrin dependence is (methyl-B-cyclodextrin) that comprises methyl B cyclodextrin, phenothiazine (phenothiazine), monodansylcadaverine, chloroquine (chloroquine), monensin (monensin) and dynasore.
3. method as claimed in claim 2, wherein the endocytosis inhibitor of clathrin dependence is dynasore.
4. a method is used for treating patient and sends the situation that causes because of reducing the transforming growth factor-beta signal, comprises the endocytosis inhibitor of the clathrin dependence that gives a treatment of patient effective dose.
5. method as claimed in claim 4, wherein the endocytosis inhibitor of clathrin dependence is (methyl-B-cyclodextrin) that comprises methyl B cyclodextrin, phenothiazine (phenothiazine), monodansylcadaverine, chloroquine (chloroquine), monensin (monensin) and dynasore.
6. method as claimed in claim 5, wherein the endocytosis inhibitor of clathrin dependence is dynasore.
7. method as claimed in claim 5, send the situation that causes because of reducing the transforming growth factor-beta signal, comprise inflammatory (inflammatory condition), cancer (cancer), cardiovascular disease (cardiovascular disease) and a skin (skin condition).
8. the method for an accelerating wound healing comprises the endocytosis inhibitor that a clathrin giving the patient treatment effective dose relies on.
9. method that is used for suppressing patient's carcinogenesis comprises the endocytosis inhibitor that a clathrin giving the patient treatment effective dose relies on.
10. an atherosis method that is used to suppress the patient comprises the dynasore that gives the patient treatment effective dose.
11. method as claimed in claim 10 also comprises the Statins inhibin (statin) that gives the patient treatment effective dose.
12. method as claimed in claim 11, wherein Statins inhibin (statin) is selected from group, it comprises lovastatin (lovastatin), simvastatin (simvastatin), pravastatin (pravastatin), fluvastatin (fluvastatin), atorvastatin (atorvastatin), itavastatin (itavastatin), rosuvastatin (rosuvastatin) and rivastatin (rivastatin).
13. a pharmaceutical composition comprises the endocytosis inhibitor that a clathrin of a treatment effective dose relies on, inhibin and a pharmaceutically acceptable carrier of a treatment effective dose.
14. method as claimed in claim 13, wherein the endocytosis inhibitor of clathrin dependence is to comprise methyl B cyclodextrin (methyl-B-cyclodextrin) (B-CD), phenothiazine (phenothiazine), monodansylcadaverine, chloroquine (chloroquine), monensin (monens in), and dynasore.
15. method as claimed in claim 14, wherein the endocytosis inhibitor of clathrin dependence is dynasore.
16. method as claimed in claim 13, wherein Statins inhibin (statin) is selected from group, it comprises lovastatin (lovastatin), simvastatin (simvastatin), pravastatin (pravastatin), fluvastatin (fluvastatin), atorvastatin (atorvastatin), itavastatin (itavastatin), rosuvastatin (rosuvastatin) and rivastatin (rivastatin).
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| WO2010033507A1 (en) | 2008-09-16 | 2010-03-25 | St. Louis University | Method of enhancing tgf-beta signalling |
| WO2014063205A1 (en) * | 2012-10-26 | 2014-05-01 | The University Of Queensland | Use of endocytosis inhibitors and antibodies for cancer therapy |
| WO2014130411A1 (en) * | 2013-02-22 | 2014-08-28 | Emory University | Tgf-beta enhancing compositions for cartilage repair and methods related thereto |
| WO2022138700A1 (en) * | 2020-12-23 | 2022-06-30 | ピアス株式会社 | Atopic skin disease ameliorating agent, external agent for skin, and cosmetic |
| WO2023277761A1 (en) * | 2021-06-28 | 2023-01-05 | Gunnar Pejler | New use of monensin |
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